A vast majority of oligonucleotide syntheses are carried out on supports to which a first nucleoside has been pre-attached (e.g., by a succinate or hydroquinone-O,O′-diacetate linker). After routine cleavage and deprotection steps, this nucleoside becomes the 3′ terminal nucleoside of the target oligonucleotide. For standard DNA and RNA synthesis, this requires an inventory of four deoxynucleoside and four ribonucleoside supports, i.e., a different support depending on the desired terminal nucleoside.
A “universal” solid support (i.e., a support without the first nucleoside attached) for DNA and RNA synthesis that permitted direct coupling of any residue and elimination of the terminal phosphodiester linkage at the same time as the deprotection step would offer several advantages. For example, such a universal support would: (a) eliminate the need for an inventory of several different nucleoside supports; (b) minimize the possibility of error in the selection of the correct support type; (c) reduce the time and eliminate errors in the generation of an array of nucleoside supports in 96 well synthesizers; and (d) allow the preparation of oligonucleotides containing a 3′-terminal nucleoside which is not available as a support.
In spite of these major potential benefits of universal supports, at the present time, the use of universal supports for DNA and RNA synthesis has not been favored. One major hurdle to overcome relates to finding conditions to eliminate the terminal phosphate, produced from the first nucleoside phosphoramidite addition cycle to the required terminal hydroxyl group. This problem is considered in more detail below.
Certain known universal supports based on the ribonucleoside elimination model are known as “McLean supports.” See S. Scott, P. Hardy, R. C. Sheppard, and M. J. McLean, “A Universal Support for Oligonucleotide Synthesis,” Innovation and Perspectives in Solid Phase Synthesis, 3rd International Symposium, 1994, pp. 115-124, which document is entirely incorporated herein by reference. McLean supports also are described in U.S. Pat. Nos. 5,681,945 and 5,886,193, which patents are entirely incorporated herein by reference. A McLean support also is illustrated in FIG. 1a. This solid support allows the detritylation, the addition of the first nucleoside monomer, and the remainder of the oligomer preparation to proceed without any changes from standard protocols. Elimination of the terminal phosphodiester group utilizes the same reagents needed for routine deprotection of oligonucleotides, but requires more aggressive and lengthy treatment conditions (e.g., concentrated ammonium hydroxide/80° C./17 hours as compared to the standard oligomer deprotection conditions of ammonium hydroxide/55° C./5-6 hours). While these more aggressive conditions are suitable for the preparation of unmodified oligonucleotides, they are not compatible with base-labile nucleoside analogues. Furthermore, prolonged treatment with basic volatile reagents or the need to employ a desalting step in the case of sodium hydroxide make this solid support unattractive for use in industrial multi-well synthesizers.
The instability of RNA to strongly basic conditions is caused by the proximity of the 2′-OH group to the phosphodiester group. Attack of the 2′-OH on the adjacent phosphorus gives rise to an energetically favorable 5-membered transition state, which can open up again to form a mixture of 2′- and 3′-phosphodiester internucleotide linkages, or can lead to chain scission by elimination of the 3′- or 5′-hydroxyl group. In the case of a McLean universal support of FIG. 1a, cleavage from the support by hydrolysis of a succinate or, better, a hydroquinone-O,O′-diacetate (O) linkage, generates a hydroxyl group adjacent to the terminal phosphodiester linkage. Additional base treatment leads to the elimination of the terminal phosphate group and formation of the desired 3′-OH. Wengel and coworkers described a similar strategy using a neighboring hydroxyl group to facilitate elimination. See C. Scheuer-Larsen, C. Rosenbohm, T. J. D. Jorgensen, and J. Wengel, “Introduction of a Universal Solid Support for Oligonucleotide Synthesis,” Nucleosides & Nucleotides, 1997, Vol. 16, pp. 67-80, which article is entirely incorporated herein by reference. Lyttle and a group at Biosearch Technologies described a variation of the Wengel procedure. The Lyttle procedure used a linkage to a polymeric support that is not hydrolyzed by base, so that extended base treatment releases the dephosphorylated oligo, while leaving any undesired by-product still attached to the support. See M. H. Lyttle, D. J. Dick, D. Hudson, and R. M. Cook, “A Phosphate Bound Universal Linker for DNA Synthesis,” Nucleosides & Nucleotides, 1999, Vol. 18, pp. 1809-1824, which article is entirely incorporated herein by reference.
The main impediment to adoption of a universal support has been the aggressively basic conditions required to complete the elimination reaction to release the terminal hydroxyl group. The standard reagents used in oligonucleotide deprotection are ammonium hydroxide and aqueous methylamine, which are popular because they are readily available and completely volatile. Using these reagents to carry out the elimination reaction, however, requires either high temperature, with attendant high pressure, or extended reaction times. The situation can be improved by adding metal ions to the mix (such as Li+, Na+, and Zn2+), to speed up the elimination reaction, presumably by stabilizing the 5-membered transition state. However, the speed and simplicity of evaporation of the deprotection solution to give the crude oligonucleotide without desalting is not possible when these ionic additives are used.
Other known universal supports that use neighboring aminomethyl or diamino-ethyl groups to assist the elimination reaction have been described by Azhayev. See A. V. Azhayev, “A New Universal Solid Support for Oligonucleotide Synthesis,” Tetrahedron, 1999, Vol. 55, pp. 787-800, which article is entirely incorporated herein by reference. These supports, which are illustrated in FIGS. 1b, 1c, and 1d, are not only compatible with the preparation of all common types of oligonucleotides, but they also function well for oligomers with unusual base labile nucleoside units. Using volatile ammonium hydroxide or aqueous methylamine with these supports (e.g., concentrated ammonium hydroxide/80° C./2-8 hours), 3′-terminal dephosphorylation was significantly speeded up, and dephosphorylation could be achieved under neutral conditions using aqueous zinc chloride or water. In these supports, the neighboring aminomethyl- or diaminoethyl-groups assist for the elimination of the terminal phosphodiester to generate termini with 3′-hydroxyl.
While the universal supports of FIGS. 1b-1d offer genuine advantages, especially if the oligonucleotides contain base-labile components, they still are not ideal for mainstream applications. These supports still require relatively long treatment times with basic volatile reagents at elevated temperatures. These features make these supports somewhat unattractive for industrial applications.
Accordingly, there is a need in the art for a universal support suitable for DNA and RNA synthesis that eliminates the drawbacks mentioned above. A desirable universal support would allow fast cleavage and dephosphorylation at relatively low temperatures (e.g., in 20 minutes at room temperature), and under relatively mild reaction conditions (e.g., using a 2M ammonia in methanol cleavage reagent). Moreover, the desired universal support would be compatible for use under ultra-mild, normal, and ultra-fast deprotection conditions. Finally, the desired universal support product will be cost-effective (e.g., comparable in price to regular 2′-deoxynucleoside supports).